The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca2+, and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca2+-release channel inositol 1,4,5-trisphosphate receptor (IP3R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP3R on the ER or mitochondrial surface, which directly enhanced Ca2+ accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP3R and the mitochondrial Ca2+ uptake machinery. Because organelle Ca2+ homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

The P2X 7 receptor is known for the cytotoxic activity because of its ability to cause opening of non-selective pores in the plasma membrane and activate apoptotic caspases. A key factor of P2X 7 -dependent cytotoxicity is the massive intracellular Ca 2؉ increase triggered by its activation. Here we show that P2X 7 transfection increased the ability of the endoplasmic reticulum to accumulate, store, and release Ca 2؉ . This caused a larger agonist-stimulated increase in cytosol and mitochondrial Ca 2؉ in P2X 7 transfectants than in mock transfected cells. P2X 7 transfectants survived and even proliferated in serum-free conditions and were resistant to apoptosis triggered by ceramide, staurosporin, or intracellular Zn 2؉ chelation. Finally, the nuclear factor of activated T cells complex 1 (NFATc1) was strongly activated in the P2X 7 transfectants. These observations support our previous finding that the P2X 7 receptor under tonic conditions of stimulation, i.e. those observed in response to basal ATP release, has an anti-apoptotic or even growth promoting rather than cytotoxic activity.Cell responses to extracellular ATP are mediated by P2 receptors: ionotropic P2X and metabotropic P2Y (1). The P2X 7 receptor (P2X 7 R) 4 subtype stands out in the P2X subfamily for its ability to trigger a host of physiologic or pathologic responses: plasma membrane blebbing (2, 3), rapid release of interleukin-1␤ via microvesicle shedding (2, 4), cell fusion (5), lymphoid cell proliferation (6), cell death (7,8), and bone formation/ resorption (9). This receptor is characterized by low affinity for ATP and by two states of permeability (10, 11). At high micromolar ATP concentrations, P2X 7 behaves as a cation-selective channel, whereas a prolonged exposure to nearly millimolar concentrations triggers the transition to a nonselective pore that admits hydrophilic solutes of molecular mass up to 900 Da (12-14). A common feature of both conductance states is a strong elevation of free cytoplasmic calcium levels ([Ca 2ϩ ] i ), a response critical for the biological role of this receptor.Recent evidence from our group shows that basal levels of ATP, naturally present in the extracellular milieu, cause a tonic activation of the P2X 7 R, which in turn triggers [Ca 2ϩ ] i increase and Ca 2ϩ entry into the mitochondria. The increased intramitochondrial Ca 2ϩ concentration ([Ca 2ϩ ] mt ) then enhances mitochondrial potential, increases ATP synthesis, and promotes survival and proliferation in the absence of serum (15). Increased mitochondrial potential and ability to grow under serum-free conditions are hallmark of tumor transformation (16). Furthermore, overexpression of the P2X 7 R is associated with several cancers or growth disturbances such as chronic lymphocytic leukemia (17), prostate (18)

Malignant hyperthemia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1␤ after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1␤ from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1␤ production. Malignant hyperthermia (MH)1 is a pharmacogenetic disease triggered by volatile anesthetics and the depolarizing muscle relaxant succinylcholine in predisposed individuals (1-4). The clinical signs of an impending MH reaction are highly variable and are caused by a hypermetabolic state with muscle rigidity, metabolic acidosis, rhabdomyolysis, tachycardia, and/or an increase in body temperature (5). In some individuals MH reactions appear to be triggered by physical exercise or emotional stress. The latter observation has led to the suggestion that MH, heat stroke, and exercise-induced rhabdomyolysis might have a common denominator (2, 6, 7). The underlying causes of MH are abnormalities in the skeletal muscle calcium metabolism (8, 9) and molecular genetic studies have mapped the primary locus of MH to chromosome 19q, the gene encoding the ryanodine receptor calcium release channel (RYR1) (2, 4, 10). Approximately 50% of MH families have mutations in the RYR1 gene, and mutations have been reported in other loci (for recent reviews, see Refs. 11 and 12).The ryanodine receptors are large tetrameric oligomers that function as intracellular calcium release channels. Three different isoforms have been identified at the molecular level: type 1 (RYR1), which is preferentially expressed in skeletal muscle; type 2, which is in the heart and cerebellum; and type 3, which is in the central nervous system as well as in a variety of other tissues (13-16). RYR1 can be pharmacologically activated by a number of compounds, among which are caffeine, halothane, thymol, 4-chloro-m-cresol, E218, bastadin, polylysine, and calcium (17-21). Activation causes the channel to open and thus to a transient calcium flow from the sarcoplasmic reticulum,...

No abstract

Junctate is an integral sarco(endo)plasmic reticulum protein expressed in many tissues including heart and skeletal muscle. Because of its localization and biochemical characteristics, junctate is deemed to participate in the regulation of the intracellular Ca 2þ concentration. However, its physiological function in muscle cells has not been investigated yet. In this study we examined the effects of junctate over-expression by generating a transgenic mouse model which over-expresses junctate in skeletal muscle. Our results demonstrate that junctate over-expression induced a significant increase in SR Ca 2þ storage capacity which was paralleled by an increased 4-chloro-mcresol and caffeine-induced Ca 2þ release, whereas it did not affect SR Ca 2þ -dependent ATPase activity and SR Ca 2þ loading rates. In addition, junctate over-expression did not affect the expression levels of SR Ca 2þ binding proteins such as calsequestrin, calreticulin and sarcalumenin. These findings suggest that junctate over-expression is associated with an increase in the SR Ca 2þ storage capacity and releasable Ca 2þ content and support a physiological role for junctate in intracellular Ca 2þ homeostasis.